Vol.4, No.

2 (2011), 289-294
ISSN: 0974-1496
CODEN: RJCABP
http://www.rasayanjournal.com

SYNTHESIS AND CYTOTOXIC, ANTI OXIDANT ACTIVITES

OF NEW CHALCONE DERIVATIVES
Mohd. Rayees Ahmed*1, V. Girija Sastry2, Nasreen Bano3, S. Ravichandra4
and M. Raghavendra5
*1,2
Department of Pharmaceutical Chemistry, University College of Pharmaceutical Sciences,
Andhra University, Visakhapatnam Andhra Pradesh, India.
3
Department of Biotechnology, Jawaharlal Nehru Technological University, Hyderabad, Andhra
Pradesh, India.
4,5
Department of Chemistry, Fathima Institute of Pharmacy, Kadapa. Andhra Pradesh, India.
*E-mail: dr.rayeespharma@gmail.com

ABSTRACT
Chalcones belong to an important class of flavonoids, which may be prepared by Claisen reaction. They possess a
wide range of biological activities and industrial applications. Kostanecki was the first to give the term chalcone and
who did pioneering work in the synthesis of naturally coloring compounds. Cytotoxicity against tumour cell lines
may be the result of disruption of the cell cycle, inhibition of angiogenesis, interference with p53-MDM2
interaction, mitochondrial uncoupling or induction of apoptosis. Structural requirements for cytotoxic activity vary
according to the mechanisms of action. Chemoprotection by chalcones may be a consequence of their antioxidant
properties, mediated via inhibition or induction of metabolic enzymes, by an anti-invasive effect or a reduction in
nitric oxide production. Chalcones are synthesized by conventional and microwave assisted synthesis methods. By
microwave assisted synthesis, a considerable increase in the reaction rate has been observed and that too, with better
yields. The compounds have been screened for cytotoxic activity and antioxidant activity.
Key words: Claisen-Schmidt condensation, Microwave irradiation, Cytotoxic activity, Antioxidant activity.
2011 RASYAN. All rights reserved.

INTRODUCTION
Chalcones are abundantly present in nature from ferns to higher plants l. They are aromatic compounds
with an unsaturated side chain and are often cytotoxic in vitro 2. Chalcones have also been reported to be
anti-inflammatory, analgesic and antipyretie3. Some chalcones possess bactericidal, antifungal and
insecticidal activity and some of their derivatives are reported to be antimutagenic 4. Chalcones are 1,3-
diphenyl-2-propene-1-one5,6, in which two aromatic rings are linked by a three carbon , - unsaturated
carbonyl system. These are abundant in edible plants and are considered to be the precursors of
flavonoids and isoflavonoids. Chalcones are synthesized by Claisen-Schmidt condensation, which
involves cross aldol condensation of appropriate aldehydes and ketones by base catalyzed or acid
catalyzed followed by dehydration. Chalcone is a common natural pigment and one of the important
intermediate in the biosynthesis of flavonoids 7. Synthetic and naturally occurring chalcones have been
extensively studied and developed as one of the pharmaceutically important molecules. chalcone
derivatives are screened for their anti-inflammatory activity8, chemopreventive activity9,cardiovascular
disease10, anticancer activity11,cytotoxic activity12,atiprolifirative activity13,antimalarial activity14,antiviral
activity15, antiHIV activity16. Therefore, in the present investigation it has been considered worthwhile to
synthesize some new chalcone derivatives by conventional and microwave irradiation methods and
comparison between two methods.
Microwave-induced organic reaction enhancement (MORE) chemistry10 is gaining popularity as
a non-conventional technique for rapid organic synthesis. Important features of this technique are easy
access to very high temperature, good control over energy input in a reaction, higher yields and rapid
synthesis of organic compounds.

NEW CHALCONE DERIVATIVES Mohd. Rayees Ahmed et. al

 Vol.4, No.2 (2011), 289-294

The synthesized compounds were purified by recrystallization and chromatography. The

compounds were characterized by 1H NMR and IR analysis. The compounds were tested for their
cytotoxic activity and antioxidant activities by standard methods.
EXPERIMENTAL
General procedure for the synthesis of chalcones by Claisen-Schmidt condensation 17-21
Synthesis of chalcones (1-5)
(a) (Conventional). Equimolar quantities (0.001mol) of 2-acetyl-5-methyl-furan and respective
aldehydes (0.001mol) were mixed and dissolved in minimum amount (3ml) of alcohol. To this, aqueous
potassium hydroxide solution (0.003mol) was added slowly and mixed occasionally for 24 hrs, at room
temperature. Completion of the reaction was identified by observing on precoated TLC plates of Merck.
After completion of the reaction, the reaction mixture was poured into crushed ice, if necessary acidified
with dil HCl. The solid separated was filtered and dried. It was purified by recrystallization or by column
chromatography performed on silica gel (100-200 mesh, Merck) using ethylacetate and hexane mixture as
mobile phase.
(b) (MWI). Equimolar quantities (0.001mol) of 2-acetyl-5-methyl-furan and respective aldehydes
(0.001mol) were mixed and dissolved in minimum amount (3ml) of alcohol. To this, aqueous potassium
hydroxide solution (0.003mol) was added slowly and mixed. The entire reaction mixture was microwave
irradiated for about 2-6 minutes at 180 watts .
The spectral data of all synthesised compounds is mentioned below
1-(5-methylfuran-2-yl)-3-phenylprop-2-en-1-one (1) : Mol. Formula: C14H12O2 , Conventional method
Yield 61, Microwave Irradiation 74%, m.p. 124 2oC. IR (cm-1) : 3020 (C-H aromatic streching), 2924
(C-H methyl streching), 1660 (C=O), 1604 (HC=CH), 1214 (C-O-C). 1H NMR ( ppm) : 2.45 (3H, s, C-
5'-CH3), 6.22 (1H, d, J=4.2 Hz, C-4'-H),7.25 (1H, d, J=15.6 Hz, CO-CH=), 7.37-7.43(5H, m, C-2'', 3'',
4''and 5'', 6''-H), 7.64 (1H, d, J=4 Hz, C-3'-H),
3-(4-fluorophenyl)-1-(5-methylfuran-2-yl) prop-2-en-1-one (2) : Mol. Formula: C14H11FO2 ,
Conventional method Yield 65, Microwave Irradiation 74%, m.p. 92 2oC. IR (cm-1) : 1644 (C=O),
1591 (HC=CH), 1215 (C-O-C), 798 (C-F). 1H NMR ( ppm) : 2.13 (3H, s, C-5'-CH3), 7.45 (1H, d, J=4
Hz, C-4'-H),7.64 (1H, d, J=16 Hz, CO-CH=),7.70 (2H, d, J=8.4 Hz, C- 3'' and 5''-H), 7.75 (1H, d, J=4.2
Hz, C-3'-H), 7.84 (2H, d, J=8.4 Hz, C -2'' and 6''-H),8.00 (1H, d, J=16 Hz, Ar-C-H=).
3-(4-chlorophenyl)-1-(5-methylfuran-2-yl) prop-2-en-1-one (3) : Mol. Formula: C14H11Cl O2 ,
Conventional method Yield 78, Microwave Irradiation 86%, m.p. 126 2oC. IR (cm-1) : 3016 (C-H),
2924 (C-H), 1651 (C=O),1602 (HC=CH), 1212 (C-O-C), 800 (C-Cl). 1H NMR ( ppm) : 2.41 (3H, s, C-
5'-CH3), 6.25 (2H, d, J=4 Hz, C- 3' and 4'-H), 7.26 (2H, d, J=8.2 Hz, C-3'' and 5''-H),7.40 (1H, d, J=16
Hz, CO-CH=), 7.58 (2H, d, J=9.4 Hz, C-2'' and 6''-H),81 (1H, d, J=16.4 Hz, Ar-C-H=).
3-(2,4-dichlorophenyl)-1-(5-methylfuran-2-yl) prop-2-en-1-one (4) : Mol. Formula: C14H10Cl 2 O2 ,
Conventional method Yield 64, Microwave Irradiation 73 %, m.p. 120 2oC. IR (cm-1) : 3020 (C-H),
2924 (C-H), 1657 (C=O),1601 (HC=CH), 1215 (C-O-C), 798 (C-Cl). 1H NMR ( ppm) : 2.44 (3H, s, C-
5'-CH3), 6.23 (1H, d, J=4 Hz, C- 4'-H), 7.26-7.30 (2H, m, C-5'' and 6''-H), 7.35 (1H, d, J=15.6 Hz, CO-
CH=), 7.45 (1H, s, C-3''-H),7.69 (1H, d, J=8 Hz, C-3' -H), 8.16 (1H, d, J=16 Hz, Ar-C-H=).
1-(5-methylfuran-2-yl)-3-(4-nitrophenyl) prop-2-en-1-one (5) : Mol. Formula: C14H11NO4 ,
Conventional method Yield 53, Microwave Irradiation 61 %, m.p. 174 2oC. IR (cm-1) : 1657 (C=O),
1603 (HC=CH),1511 (Ar-NO2), 1213 (C-O-C). 1H NMR ( ppm) : 2.46 (3H, s, C-5'-CH3), 6.25 (1H, d,
J=4 Hz, C- 4'-H),7.30 (1H, d, J=4.2 Hz, C-3'-H),7.44 (1H, d, J=16 Hz, CO- CH= ), 7.75 (2H, d, J=9.2 Hz,
C-2'' and 6'' -H),7.81 (2H, d, J=9.4 Hz, C-3'' and 5''-H), 8.24 (1H, d, J=16.4 Hz, Ar-C-H=).

Cytotoxicity test
Brine shrimp lethality bioassy (BSLT)
Brine shrimp lethality test have been used as bioassay for a variety of toxic substances. This method has
also been applied to plant extracts in order to facilitate the isolation of biologically active compounds, 19,
20, 21
. A general bioassay that appears capable of detecting a broad spectrum of bioactivity, present in
NEW CHALCONE DERIVATIVES 290 Mohd. Rayees Ahmed et. al
 Vol.4, No.2 (2011), 289-294

crude extracts and in synthetic compounds is the brine shrimp lethality bioassay, rather than more tedious
and expensive in vitro and in vivo antitumor assays. Furthermore, it does not require animal serum as is
needed for cytotoxicities.
Procedure
Brine shrimp lethality bioassay was carried out to investigate the cytotoxicity of medicinal plants. Brine
shrimps (Artemia salina) were hatched using brine shrimp eggs in a conical shaped vessel (1L), filled
with sterile artificial sea water under constant aeration for 38 h. After hatching, active nauplii free from
egg shells were collected from brighter portion of the chamber and used for the assay. Ten nauplii were
drawn through a glass capillary and placed in each vial containing 5 ml of brine solution. In each
experiment, test substances whose activities are to be checked were added to the vial according to their
concentrations and maintained at room temperature for 24 h under the light and surviving larvae were
counted.
Experiments were conducted along with control (vehicle treated), different concentrations (1-
5000 g / ml) of the test substances in a set of three tubes per dose. Replicas should be maintained to get
accurate results.
Statistical analysis
The percentage lethality was calculated from the mean survival larvae of compounds treated tubes and
control. ED50 values were obtained by (best-fit line method) plotting a graph, taking concentration on X-
axis and percentage inhibition on Y-axis, at 50% of the percentage inhibition the line was drawn from Y-
axis and aligned with the concentration on X-axis then got the ED50 values.
Antioxidant activity
Free radicals are formed constantly in human system either as accidental products during
metabolism or deliberately during the process of phagocytosis; or due to environmental pollutants,
ionizing radiations, ozone, heavy metal poisoning, cigarette smoking and chronic alcohol intake. Free
radicals being highly reactive can oxidize biomolecules leading to tissue injury and cell death.
In the present study, two in vitro antioxidant models 1,1-Diphenyl-2-picrylhydrazyl radical
(DPPH) scavenging activity (as it is a model for lipophilic radicals which initiate lipid peroxidation) .
The IC50 values of chalcones tested for their antioxidant activity. Solvent used in both the tests for
compounds was DMSO (Dimethylsulphoxide).
DPPH free-radical scavenging activity
DPPH (1,1diphenyl-2-picrylhydrazyl) radical scavenging activity was measured by the method
of Lamaison et al. The reaction mixture contained 1.5X10-7 M methanolic solution of DPPH and various
concentrations of the test substances and were kept in dark for 50 minutes. Optical density (OD) of
samples was measured at 517 nm against a blank, and IC50 values were calculated (using linear regression
analysis) by plotting a graph, taking concentration on X-axis and percentage inhibition on Y-axis, at 50%
of the percentage inhibition the line was drawn from Y-axis and aligned with the concentration on X-axis
then got the IC50 values.
RESULTS AND DISCUSSION
In the present study, we have performed the synthesis of chalcone derivatives by conventional and
microwave irradiation method in Scheme -1 but to reduce the reaction time, it was decided to synthesize
the compounds with microwave irradiation, which can be more effective, faster, and energy efficient in
addition; we have compared those with others that were obtained via conventional heating methods and
results were mentioned in Table-1 and Table-2.
Brine shrimp lethality test have been used as bioassay for variety of toxic substances, all the
chalcones (1-5) were tested for cytotoxic activity by the BSLT bioassay method. All the compounds were
found to possess cytotoxic activity. Among them, compounds 1, 3showed dose dependent cytotoxic
activity at concentrations of (1) 24.27 g/ml, (3) 37.05 g/ml, respectively. The remaining compounds
exhibited less activity when compared to the above compounds at various concentration levels.
Podophyllotoxin is used as a standard drug for BSLT assay method. By comparing the results compound
1, 3 found to be the best among all the tested compounds. The results and complete data of test presented
in Table-3.The potency of the chalcone derivatives was estimated by ED50 values. Few of the chalcone
NEW CHALCONE DERIVATIVES 291 Mohd. Rayees Ahmed et. al
 Vol.4, No.2 (2011), 289-294

derivatives showed good percentage inhibition but their ED50 values were more. Hence they were less
potent among the tested compounds with respect to ED50 values.

O
O
Ethanol, Aq. KOH
H3C
O
C CH3 + Ar -- CHO H3C
O
Ar
Room Temperature
1-(5-methylfuran-2-yl)ethanone 12 -- 24 hours
Micro Wave Irradiation

Where, Ar = Phenyl (1), 4- Fluoro phenyl(2), 4- Chloro phenyl(3), 2,4- Dichloro phenyl(4), 4- Nitro phenyl(5)
Scheme-1

Table-1: Comparative reaction time and percentage yield of chalcone derivatives by conventional and microwave
irradiation methods.
S.No. Reaction time Yeild (%)
Conventional (hr) MW ( min) Conventional MW
1 24 3 61 74
2 24 4 65 74
3 24 3.5 78 86
4 24 3.5 64 73
5 24 4 53 61

The in vitro antioxidant activity and scavenging effects of the 5 chalcones were evaluated by using
different reactive species assay containing DPPH radical scavenging activity. The potency of the chalcone
derivatives was estimated by IC50 values. The IC50 values of chalcone derivatives used in the present study
were given in Table-4.
DPPH (1,1diphenyl-2-picrylhydrazyl) radical scavenging activity was measured for all the
chalcones (1-5). Among them, compounds 1, 2, 3, 4 and 5 showed a dose dependent inhibition of radicals
at concentrations of 25, 50 and 100 g/ml.
Ascorbic acid, the well known antioxidant was used in the test for comparing the results,
compounds 5 appears to be the best among all the tested compounds. Few of the chalcone derivatives
showed good percentage inhibition but their IC50 values were more. Hence they were less potent among
the tested compounds with respect to IC50 values.

CONCLUSION
All the synthesized (5) compounds were purified by recrystallization or by column chromatography. The
identification of compounds was established by single spot TLC, melting point and by spectral analysis
involving IR, 1H NMR, 13C NMR and elemental analysis. Since chalcones were widely reported to
possess cytotoxic and antioxidant activities etc. All the chalcone derivatives were evaluated for the above
mentioned activities and they have exhibited promising activity.
From the cytotoxic and antioxidant activities it was proven that most of the chalcone derivatives
are potent and possessing cytotoxic and antioxidant activities.

ACKNOWLEDGEMENTS
I am thankful to my guide Dr.V.Girija Sastry, Dept. of pharmaceutical chemistry, University College of
Pharmaceutical Sciences, Andhra University, Visakhapatnam - 530003 (A.P.) India. For providing the
facilities to carry out research work. To my beloved parents (Father Md. Rafeeq Ahmed and Mother
Khyrunnisa begum ). For their moral support to pursue Ph.D programme.
NEW CHALCONE DERIVATIVES 292 Mohd. Rayees Ahmed et. al
 Vol.4, No.2 (2011), 289-294

Table -2: Characterization of chalcone derivatives

Quantity (g/ml)
Compound Percentage inhibition
25 g/ml 50 g/ml 100 g/ml IC50 g/ml
1 7.24 12.31 16.03 76.12
2 4.05 7.12 11.04 65.04
3 9.11 10.03 12.04 81.15
4 8.35 9.24 10.47 75.20
5 10.14 11.85 13.69 49.18
Ascorbic 16.13 38.11 62.34 0.61
acid 1 g/ml 2.5 g/ml 5 g/ml

Table-3: Cytotoxic activity of chalcones by using Brine shrimp lethality test (Compounds 1-5)

S.NO Compound Solubility ED50 g/ml

1 Phenyl DMSO 24.27
2 4- Fluoro phenyl - 39.26
3 4- Chloro phenyl - 37.05
4 2,4- Dichloro phenyl - 43.53
5 4- Nitro phenyl - 45.38
Standard (Podophyllotoxin) - 3.88

Table-4: Percentage inhibition of free radicals using DPPH method (Compounds 1-5)
Compound Rf value M.P Elemental analysis
Calculated(%) Found(%)
1 0.62 124 2oC C: 79.17 C: 79.2
H: 5.65 H: 5.68
O: 15.08 O: 15.1
2 0.64 92 2oC C: 72.98 C: 72.95
H: 4.77 H: 4.80
O: 13.90 O: 13.87
3 0.66 126 2oC C: 68.12 C: 68.09
H: 4.46 H: 4.49
O: 12.97 O :12.94
4 0.62 120 2oC C: 59.78 C: 59.81
H: 3.55 H: 3.52
O: 11.38 O: 11.35
5 0.56 174 2oC C: 54.18 C: 54.21
H: 3.42 H: 3.45
O: 24.88 O: 24.91

REFERENCES
1. A.W. Star, and T.J. Marby, Phytochemistry, 10, 2812(1971)
2. D.N. Dhar, ,Wiley,213(1981).
3. K. Satyanarayana, and , M.N.A. Rao,.Indian Drugs, 30,313. (1993).
4. T. Torigoo, M. Arisawa, S. Iloch, M. Fujiu, and H.B. Mayuyama, Biochem. Biophys. Res.
Commun., 112, 833.
5. Nowakowska., Eur. J. Med. Chem, 42, 125 (2007).
6. S.Maayan, N. Ohad, and K. Soliman, Bioorg. Med. Chem, 13 (2), 433 (2005).
NEW CHALCONE DERIVATIVES 293 Mohd. Rayees Ahmed et. al
 Vol.4, No.2 (2011), 289-294

7. M.L .Go, X. Wu, and X.L. Liu, Current. Medicinal. Chemistry, 12, 483 (2005)
8. Young Hoon Kim, Jeongsoo Kim, Haeil Park, Hyun Pyo Kim., Biol. Pharm Bull, 30 (8), 1450-
1455, (2007).
9. W. Shen Jeu, L. Cheng Tsung, T. Lo Ti, W. Jing Ru, K. Hrogn Huey. W. Jih Pyang, and L. Chun
Nan, Eur. J. Med. Chem., 40, 103 (2005).
10. N.Liming, W.J. Kimberly, M. Weingarten, and S.A. Janes, PCT Int. Appl., 411 (2003).
11. E.Francesco, G. Salvatore, M. Luigi, and C. Massimo, Phytochem., 68, 939 (2007).
12. S. Ducki, R. Forrest, J.A. Hadfield, A. Kendall, N.J. Lawrence, A.T. Mc Gown and D. Rennson,
Bioorg. Med. Chem. Lett., 8, 1051 (1998).
13. E. Bombardelli, and P. Valenti, PCT Int. Appl., 18 (1998).
14. M.Chen, T.G. Theander, S.B .Christensen, L. Hrlid, Zhai and A. Kharazmi, Antimicrob. Agents.
Chemother, 38, 1470 (1994).
15. J.C.Onyilagha, B. Malhotra, M. Elder, and G.H.N Towers, Can., J. Plant. Pathol., 19, 133 (1997).
16. H.X .Xu, M. Wan, H. Dong, P. But, and L.Y. Foo, Biol. Pharm. Bull., 23, 1072 (2000).
17. J.H. Min, H. Jiaxing, H. Weiyi, and H. Hongmen, Ind. J. Chem., 40 B, 1222 (2001).
18. W.Dawey, and D. Tivey, J. Chem. Soc., 1320 (1958).
19. H.M.Kohler, H. Chadwell, H. Gillman, A.H. Blatt(Eds.), Organic Synthesis Wiley Interscience,
New York, 78, Coll. Vol.1 (1967).
20. H.S. Mehra, J. Ind. Chem. Soc., 45, 178 (1968).
21. M.J.Climent, A. Corma, S. Iborra, and J. Primo., J. Catalyst., 151, 60 (1995).
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